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J Biol Chem, Vol. 274, Issue 51, 36616-36622, December 17, 1999

Molecular Cloning, Sequencing, and Expression of the Gene Encoding Alkaline Ceramidase from Pseudomonas aeruginosa
CLONING OF A CERAMIDASE HOMOLOGUE FROM MYCOBACTERIUM TUBERCULOSIS^*

Nozomu Okino, Sachiyo Ichinose, Akira Omori, Shuhei Imayama^§, Takashi Nakamura, and Makoto Ito^¶

From the Department of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences and the ^§ Department of Dermatology, Faculty of Medicine, Graduate School Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan and the  Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida 194-8511, Tokyo, Japan

We previously reported the purification and characterization of a novel type of alkaline ceramidase from Pseudomonas aeruginosa strain AN17 (Okino, N., Tani, M., Imayama, S., and Ito, M. (1998) J. Biol. Chem. 273, 14368-14373). Here, we report the molecular cloning, sequencing, and expression of the gene encoding the ceramidase of this strain. Specific oligonucleotide primers were synthesized using the peptide sequences of the purified ceramidase obtained by digestion with lysylendopeptidase and used for polymerase chain reaction. DNA fragments thus amplified were used as probes to clone the gene encoding the ceramidase from a genomic library of strain AN17. The open reading frame of 2,010 nucleotides encoded a polypeptide of 670 amino acids including a signal sequence of 24 residues, 64 residues of which matched the amino acid sequence determined for the purified enzyme. The molecular weight of the mature enzyme was estimated to be 70,767 from the deduced amino acid sequence. Expression of the ceramidase gene in Escherichia coli, resulted in production of a soluble enzyme with the identical N-terminal amino acid sequence. Recombinant ceramidase was purified to homogeneity from the lysate of E. coli cells and confirmed to be identical to the Pseudomonas enzyme in its specificity and other enzymatic properties. No significant sequence similarities were found in other known functional proteins including human acid ceramidase. However, we found a sequence homologous to the ceramidase in hypothetical proteins encoded in Mycobacterium tuberculosis, Dictyostelium discoideum, and Arabidopsis thaliana. The homologue of the ceramidase gene was thus cloned from an M. tuberculosis cosmid and expressed in E. coli, and the gene was demonstrated to encode an alkaline ceramidase. This is the first report for the cloning of an alkaline ceramidase.

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^* This work was supported in part by a Grant-in Aid for Scientific Research (B) (09460051) from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AB028646.

^¶ To whom correspondence should be addressed: Laboratory of Marine Biological Chemistry, Dept. of Bioscience and Biotechnology, Division of Bioresource and Bioenvironmental Sciences, Graduate School Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812-8581, Japan. Tel.: 81-92-642-2900; Fax: 81-92-642-2900 or 81-92-642-2907; E-mail: makotoi@agr.kyushu-u.ac.jp.

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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.

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